不同表面处理钛片对成骨细胞骨架影响的研究

目的研究喷砂、酸蚀、碱热处理的钛片表面对成骨细胞F- actin骨架的影响。方法将纯钛钛片根据处理方法不同分为6组：机械打磨组（ G组）、喷砂组（ SB组）、酸蚀组（ SLA组）、光滑碱热组（ AH1组）、喷砂碱热组（ AH2组）、喷砂酸蚀碱热组（ AH3组）。在各组钛片表面培养成骨细胞1、2、4、12 h后,用Phalloidin- TRITC染色,在激光共聚焦显微镜下观察6组钛片表面成骨细胞F- actin骨架的变化。结果成骨细胞接种于6组钛片表面1 h后,各组表面肌动蛋白纤维丝不能清晰看见,SB、AH2、AH3、SLA组肌动蛋白呈环状,纤维分布于粗糙表面隆起部分的边缘。接种2 h后,各组肌动蛋白纤维开始铺展,SB组微丝束有方向性的排列;G组细胞的肌动蛋白纤维成束,排列于细胞周围,有沿沟纹方向铺展趋势;AH1组细胞肌动蛋白纤维于核周围的环绕核呈平行环状排列,周边纤维呈放射状分布;AH2、AH3组纤维呈网状排列,周边纤维汇集呈指状突起,伸入周围孔洞或附着于表面隆起部分的边缘。接种4 h后,G、AH1组肌动蛋白纤维开始定向平行排列;与细胞长轴一致。接种12 h后,各组表面肌动蛋白纤维完全铺展,互相平行排列,与细胞长轴一致,并横跨整个细胞,止于细胞周缘。SB、AH3、SLA组细胞肌动蛋白纤维汇集成束,伸入周围孔洞或附着于表面隆起部分的边缘,将细胞悬挂于凹窝上方。结论成骨细胞接种于6组钛片后,在不同的表面上肌动蛋白的重组有一定的顺序和形态。SB表面最利于细胞骨架排列及伸展。     

TiO2喷砂酸蚀处理对钛片表面氧化膜及成骨细胞生长影响的研究

目的研究纯钛钛片经喷砂及喷砂酸蚀处理后,表面氧化膜金相结构和化学成分的变化及对成骨细胞黏附和生长特性的影响。方法将直径为15 mm、厚度为1 mm的纯钛钛片分4组进行表面处理：1）机械打磨组（S0）;2）喷砂组（SB）;3）喷砂酸蚀1组（SLA1）;4）喷砂酸蚀2组（SLA2）。采用电子探针分析仪及X射线衍射仪检测4组钛片表面氧化膜的厚度、化学成分以及金相结构,扫描电镜观察其表面微观形态。而后将成骨细胞培养于4组钛片表面,采用MTT法分析比较4组钛片表面对成骨细胞黏附率以及增殖率的影响。结果与S0组相比,SB、SLA1、SLA2组的粗糙度明显增大（P<0.05）。SB、SLA1、SLA2组间表面平均粗糙度差异无统计学意义（P>0.05）。酸蚀处理使喷砂形成的氧化膜变薄,密度减低,且结构发生改变：原有的金红石型TiO2峰消失,锐钛矿型TiO2减少。在表面平均粗糙度相同条件下,SB组钛片表面氧化膜均匀致密,有利于成骨细胞早期的黏附和增殖。结论喷砂和喷砂酸蚀处理均增加了钛片表面的粗糙度,有利于成骨细胞的黏附和增殖,但酸蚀使TiO2喷砂表面的氧化膜层变薄,在平均粗糙度不变的情况下,单纯喷砂表面成骨细胞的黏附和增殖优于喷砂酸蚀处理表面。     

Effect of bioactive glass air-abrasion on the wettability and osteoblast proliferation on sandblasted and acid-etched titanium surfaces

The aim of this study was to evaluate the hydrophilicity, surface free energy, and proliferation and viability of human osteoblast-like MC3T3-E1 cells on sandblasted and acid-etched titanium surfaces after air-abrasion with 45S5 bioactive glass, zinc-containing bioactive glass, or inert glass. Sandblasted and acid-etched titanium discs were subjected to air-abrasion with 45S5 bioactive glass, experimental bioactive glass (Zn4), or inert glass. Water contact angles and surface free energy were evaluated. The surfaces were studied with preosteoblastic MC3T3-E1 cells. Air-abrasion with either type of glass significantly enhanced the hydrophilicity and surface free energy of the sandblasted and acid-etched titanium discs. The MC3T3-E1 cell number was higher for substrates air-abraded with Zn4 bioactive glass and similar to that observed on borosilicate coverslips (controls). Confocal laser scanning microscopy images showed that MC3T3-E1 cells did not spread as extensively on the sandblasted and acid-etched and bioactive glass-abraded surfaces as they did on control surfaces. However, for 45S5- and Zn4-treated samples, the cells spread most at the 24 h time point and changed their morphology to more spindle-like when cultured further. Air-abrasion with bioactive glass and inert glass was shown to have a significant effect on the wettability and surface free energy of the surfaces under investigation. Osteoblast cell proliferation on sandblasted and acid-etched titanium discs was enhanced by air-abrasion with 45S5 bioactive glass and experimental Zn4 bioactive glass compared with air-abrasion with inert glass or no air-abrasion.     

微弧氧化钛表面对成骨细胞形态及细胞骨架的影响

目的 研究微弧氧化钛表面对成骨细胞形态及细胞骨架的影响。方法 将直径15 mm、厚度1 mm的纯钛片根据表面处理方法不同分为4组：机械打磨（G）组、喷砂（SB）组、打磨微弧氧化（GMAO）组和喷砂微弧氧化（SBMAO）组。采用扫描电子显微镜（SEM）、激光共聚焦显微镜（LSCM）研究钛片表面成骨细胞生长情况及细胞骨架的改变。结果 成骨细胞接种12 h后,各组细胞均沿钛片表面铺展开,且GMAO组和SBMAO组细胞覆盖于火山口状微孔上。各组肌动蛋白纤维清晰可见,GMAO组和SBMAO组肌动蛋白纤维平行排列,汇聚成束伸向微孔内。结论 微弧氧化后的钛表面可以影响成骨细胞铺展的形态及细胞骨架的排列。     

Protein adsorption and osteoblast responses to heat-treated titanium surfaces

The clinical success of dental implants is governed in part by surface properties of implants and their interactions with the surrounding tissues. The objective of this study was to investigate the effect of heat-treated titanium (Ti) surfaces on protein adsorption and osteoblast responses in vitro. The passivated Ti samples used in this study were either nonheat-treated or heat-treated at 750 degrees C for 90 minutes. Using x-ray diffraction analyses, no oxide peaks were observed on the nonheat-treated surfaces, suggesting an amorphous oxide. Crystalline rutile TiO2 peaks were observed on the heat-treated Ti surfaces. The contact angles of water on heat-treated Ti surfaces (32.0 +/- 2.5 degrees) were statistically lower compared with the nonheat-treated Ti surfaces (47.7 +/- 2.3 degrees). In addition, the mean albumin concentration on the nonheat-treated Ti surfaces (3.57 +/- 0.33 micrograms/mL) was observed to be significantly different from the mean albumin concentration on heat-treated Ti surfaces (2.25 +/- 0.26 micrograms/mL). In the presence of an osteoblast precursor cell line, significantly different hexosaminidase activity, protein production, and alkaline phosphatase activity were observed for cells grown on heat-treated Ti surfaces compared with nonheat-treated Ti surfaces.     

中药桂皮醛对成骨细胞增殖及成骨功能的影响

牙周炎和种植体周围炎是牙菌斑引起的发生在牙周支持组织上的慢性感染性炎症,通常可导致牙周组织结构破坏,引起牙和种植体松动脱落。课题组前期研究发现中药桂皮醛能杀灭牙周可疑致病菌并具有良好的抗炎作用。目的：研究桂皮醛对成骨细胞增殖及成骨功能的影响。方法：以小鼠成骨细胞MG63细胞株为研究对象,通过MTT法检测128,64,32,16,8ugml-1五种浓度的桂皮醛对成骨细胞增殖的影响;通过碱性磷酸酶活性、钙含量的测定及矿化结节的观察等检测五种浓度的桂皮醛对成骨细胞成骨功能的影响。利用SPSS13.0软件对数据进行方差分析和t检验。结果：与空白对照组相比,16,8ugml-1组成骨细胞的增殖无明显抑制,碱性磷酸酶活性、钙含量及矿化结节数有升高（P〈0.05）;128,64,32ugml-1组成骨细胞增殖及成骨功能均有一定抑制,且抑制作用随着浓度升高而加强。结论：桂皮醛对成骨细胞增殖及成骨功能的影响与浓度有关,体现出效应剂量依赖性。较低浓度的桂皮醛（16,8ugml-1）对成骨细胞的成骨功能有促进作用。     

Formation of mineralizing osteoblast cultures on machined, titanium oxide grit-blasted, and plasma-sprayed titanium surfaces

Altering osseous responses at implant surfaces to enhance bone is a current goal of clinical therapy. Cell culture may be used to investigate surface-dependent responses of bone-forming cells. In this report, the ability of primary fetal bovine mandibular osteoblast cultures to form a mineralizing matrix on machined, titanium plasma-sprayed, and titanium oxide grit-blasted surfaces has been compared. Immunohistochemical markers associated with bone formation were used to define the differentiated state of the formed matrix using qualitative light microscopy, and von Kossa staining was used to demonstrate the presence of mineralization within this matrix. Compared to either titanium oxide grit-blasted or machined surfaces, titanium plasma-sprayed surfaces displayed a unique pattern of mineralized matrix formation. Scanning electron microscopy further revealed that each surface accumulated unique organic and inorganic deposits during matrix formation, suggesting that surface-dependent physicochemical and biochemical conditioning of implant surfaces takes place. Surface topographic features of commercially pure titanium substrates can alter cultured osteoblast extracellular matrix formation and mineralization. Similar molecular and cellular assessment of in vivo responses to implant surface topography may contribute to improved engineering of endosseous implants.     

Preparation and characterization of anodized titanium surfaces and their effect on osteoblast responses

In this study, titanium (Ti) surface was modified by anodizing with a mixture of beta-glycerophosphate sodium and calcium (Ca) acetate, and the anodized surfaces were characterized by scanning electron microscopy, X-ray diffraction, and electron probe microanalysis. In vitro osteoblast response to anodized oxide was also evaluated. The anodic oxide produced was observed to have interconnected pores (0.5-2 microm in diameter) and intermediate roughness (0.60-1.00 microm). In addition, anodic oxide was observed to have amorphous and anatase oxide. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Osteoblast differentiation, as indicated by alkaline phosphatase production, was enhanced on anodized surfaces. It was thus concluded from this study that Ca phosphate can be deposited on Ti surfaces by anodization. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of Ca phosphate and higher roughness on anodized Ti surfaces.     

Early and immediate loading of titanium implants with fluoride-modified surfaces: results of 5-year prospective study

Objective: Multiple experimental and animal studies have shown that topographic, mechanical and chemical properties of implant surfaces lead to in vivo responses such as increased bone formation, increased bone anchorage and reduced healing time. A fluoride modification of the titanium implant surface also seems to positively influence bone anchorage as compared with unmodified titanium implants. Using implant survival and marginal bone loss as primary outcome parameters, the purpose of the present prospective study was to investigate whether a fluoride modification of the titanium implant surface has positive clinical effects. Materials and methods: The 17 patients included in this study received 49 Astra Tech OsseoSpeed^? implants for various indications in the maxilla and mandible. Implants were either loaded immediately or after a mean healing period of 9.56 weeks. Fifteen patients were followed up clinically including radiographic examination for 5 years. Forty‐two implants were assessed for implant survival, marginal bone loss, surgical and/or prosthetic complications, presence or absence of plaque, signs of inflammation and size of the papilla. Results: Of the original 17 patients, 15 patients were available for the full 60‐month follow‐up. One early implant failure occurred, leading to an implant survival rate of 97%. Radiographic analyses demonstrated stable bone conditions with a mean marginal bone loss of 0.1 mm (SD 0.4 mm, min ?0.7 mm, max 1.7 mm) after 5 years of function. Immediately loaded implants did not show a different mean marginal bone loss as compared with implants that were not loaded immediately. Repeated soft‐tissue examinations revealed healthy conditions in terms of 6.1% plaque and 4.2% of the implants with signs of inflammation at the 5‐year control. Discussion: Implants used in this study had high survival and success rates after 5 years. Marginal bone was well maintained, irrespective of the loading regime. To cite this article:
Mertens C, Steveling HG. Early and immediate loading of titanium implants with fluoride‐modified surfaces: results of 5‐year prospective study.
Clin. Oral Impl. Res. xx , 2011; 000–000.     

Improved retention and bone-tolmplant contact with fluoride-modified titanium implants

PURPOSE: The purpose of the present study was to investigate whether a fluoride modification of the titanium surface would have an effect on bone response after implantation. MATERIALS AND METHODS: Titanium-oxide-blasted titanium implants with and without fluoride modification were investigated in a rabbit tibia model. Quantitative analysis of surface roughness, biomechanical interlocking, and in vivo tissue reactions in rabbit bone at 1 and 3 months after placement were compared. RESULTS: The fluoride-modified test implants had a slightly smoother surface (Sa: 0.91 +/- 0.14 microm) than the unmodified control implants (Sa: 1.12 +/- 0.24 microm). Significantly higher removal torque values (85 +/- 16 Ncm vs 54 +/- 12 Ncm) and shear strength between bone and implants (23 +/- 9 N/mm2 vs 15 +/- 5 N/mm2) were measured for the fluoride-modified implants after 3 months. The histomorphometric evaluations demonstrated higher bone-to-implant contact for test implants at 1 month (35% +/- 14% vs 26% +/- 8%) and 3 months (39% +/- 11% vs 31% +/- 6%) after placement. DISCUSSION: Implant surface modification with fluoride may result in morphologic and physiochemical phenomena that are of significance for the bone response. Another possible explanation for the findings in the present study is that a surface modification changes the surface chemical structures to be more suitable for bone bonding. CONCLUSION: Based on the biomechanical and histomorphometric data, the fluoride-modified titanium implants demonstrated a firmer bone anchorage than the unmodified titanium implants. These implants achieved greater bone integration than unmodified titanium implants after a shorter healing time.     

Effect of chemically modified titanium surfaces on protein adsorption and osteoblast precursor cell behavior

PURPOSE: To investigate the effects of different chemically modified titanium surfaces on protein adsorption and the osteoblastic differentiation of human embryonic palatal mesenchymal (HEPM) cells. MATERIALS AND METHODS: Three different surfaces were evaluated. The first, a machined surface (Ti-M), was considered a control. The second surface was acid etched (Ti-AE). The third surface was prepared by exposing the Ti-AE samples to sodium hydroxide (NaOH) solution (Ti-AAE). The surface characteristics of chemically modified titanium were investigated by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and profilometry. To evaluate the production of biomarkers, commercial kits were utilized. RESULTS: Surface composition and morphology affected the kinetics of protein adsorption. Ti-AE surfaces manifested a greater affinity for fibronectin adsorption compared to Ti-M or Ti-AAE surfaces. It was observed that Ti-AE and Ti-AAE surfaces promoted significantly greater cell attachment compared to Ti-M surfaces. Statistically significant differences were also observed in the expression of alkaline phosphatase (ALP) activity, osteocalcin, and osteopontin on all 3 titanium surfaces. ALP activity and osteocalcin production up to day 12 suggested that differentiation of the cells into osteoblasts had occurred and that cells were expressing a bone-forming phenotype. CONCLUSIONS: It was thus concluded from this study that surface morphology and composition play a critical role in enhancing HEPM cell proliferation and differentiation into osteoblast cells.     

Effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell initial attachment

The clinical success of dental implants is governed in part by surface properties of implants and their interactions with the surrounding tissues. The objective of this study was to investigate the effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell attachment in vitro. Passivated titanium samples used in this study were either non heat treated or heat treated at 750 degrees C for 90 minutes. It was observed that the contact angle on heat-treated titanium surfaces was statistically lower compared with the non-heat-treated titanium surfaces. The non-heat-treated titanium surface was also observed to be amorphous oxide, whereas heat treatment of titanium resulted in the conversion of amorphous oxide to crystalline anatase oxide. No significant difference in albumin and fibronectin adsorption was observed between the heat-treated and non-heat-treated titanium surfaces. In addition, no significant difference in initial cell attachment was observed between the two groups. It was concluded that heat treatment of titanium resulted in significantly more hydrophilic surfaces compared to non-heat-treated titanium surfaces. However, differences in oxide crystallinity and wettability were not observed to affect protein adsorption and initial osteoblast precursor cell attachment.     

促矿化液对大鼠成骨细胞增殖分化的影响

目的比较大鼠成骨细胞在矿化条件与非矿化条件下增殖和分化的过程,评价促矿化液对其生理功能的影响,明确促矿化液加入细胞培养环境的适宜时间。方法取SD大鼠头盖骨做成骨细胞原代培养,将增殖稳定后的第4代细胞分别在矿化条件和非矿化条件下培养,检测其形态、碱性磷酸酶活性、细胞周期等。结果大鼠成骨细胞增殖基本稳定后促矿化液组与无促矿化液组细胞分裂增殖指数相似,但前者碱性磷酸酶活性明显较高且持久。结论成骨细胞增殖基本稳定后加入促矿化液对细胞增殖无影响,但可明显促进细胞矿化功能,是加入促矿化液诱导细胞矿化功能的较好时机。     

Emdogain promotes osteoblast proliferation and differentiation and stimulates osteoprotegerin expression

PURPOSE: The purpose of this study was to investigate the effects of EMD on the growth and differentiation of osteoblastic cells (MC3T3-E1) and on the expression of osteoprotegerin (OPG), a key cytokine that inhibits osteoclastogenesis and osteoclast function. STUDY DESIGN: MC3T3-E1 cells were treated with 100 microg/mL EMD in serum-free medium for 1, 2, 3, 5, and 7 days, or in 2% fetal bovine serum (FBS) for 3 weeks. Cells incubated without EMD served as negative control. At the end of each incubation period, cell numbers were counted and total cellular mRNA was extracted. Northern blot analysis and RT-PCR were performed to determine the mRNA levels of core binding factor alpha (Cbfa1), collagen alpha1 (I), bone sialoprotein (BSP), osteocalcin (OC), insulin-like growth factor I (IGF-I), and OPG. Alkaline phosphatase (ALP) activity was also determined and compared between treatment and control groups. RESULTS: A marked increase in cell numbers was observed in EMD-treated groups from day 2 to day 7 (P < .01). mRNA expression of collagen alpha1 (I), BSP, OC, OPG, and IGF-I were up-regulated in cells treated with EMD. ALP activity was significantly increased by EMD treatment after 3-week culture under differentiating conditions (P < .05). The expression of Cbfa1 was not affected by EMD treatment from day 1 to day 5; the levels were elevated after culturing for 3 weeks in EMD-treated cells. CONCLUSIONS: EMD promotes both proliferation and differentiation of MC3T3-E1 cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.     

导电高分子聚吡咯纯钛表面改性对成骨细胞生长、增殖和功能分化的影响

目的 研究电场刺激下纯钛（Ti）表面聚吡咯（Ppy）涂层对成骨细胞的生长、增殖和功能分化的影响。方法 成骨细胞接种于材料表面，施加100mV阳极电刺激。采用免疫荧光染色、碱性磷酸酶（APL）活性和骨钙素（OC）合成检测法观察成骨细胞在Ppy涂层表面的生长和功能表达。结果 成骨细胞在Ti以及Ti表面聚吡咯涂层的基底上均能良好生长，培养至第7天、14天、21天、28天时，阳极电刺激组（Ti Ppy＋电刺激，Ti 电刺激）的ALP活性以及OC分泌量均明显高于无电刺激组(Ti Ppy,Ti）（P＜0.05）；并且在第7天、14天、21天时，Ti Ppy 电刺激组的ALP活性以及OC分泌量又明显高于Ti＋电刺激组（P＜0.01）。结论 聚吡咯涂层具有良好的生物相容性，并且在阳极电刺激作用下对成骨细胞的增殖和分化有明显的促进作用。     

Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.